Assessing health status of ruditapes philippinarum exposed to caffeine, carbamazepine, ibuprofen and novobiocin using the neutral red retention assay

Despite the fact that pharmaceuticals have been detected in the environment at the µg L-1 to ng L-1 range, it has been demonstrated they can cause adverse effects at environmentally relevant concentrations. To detect possible adverse effects of a contaminant in the environment, standardized short-term, sensitive and low-cost methods are usually applied to estimate chronic toxicity against organisms. Lysosomal membrane stability (LMS), has been successfully applied as a screening tool to determine the health status of a wide range of organisms. The feeding style and habitat of the manila clam (Ruditapes philippinarum) make this species vulnerable to organic contaminants bound to water and it has been widely used in ecotoxicological studies. LMS was evaluated in clams’ haemolymph by the neutral red retention assay (NRRA). Clams were exposed in the laboratory to concentrations of caffeine (psychoactive stimulant), carbamazepine (anticonvulsant and mood stabilizing), ibuprofen (non-steroidal anti-inflammatory) and novobiocin (antibiotic) during 35 days in a semi-static 48 h renewal assay. Filtered sea water was spiked every 2 days with caffeine (0.1, 5, 15, 50 µg L-1), ibuprofen (0.1, 5, 10, 50 µg L-1), carbamazepine and novobiocin (0.1, 1, 10, 50 µg L-1). Stock solutions of pharmaceuticals were prepared in DMSO (0.001%) to ensure solubility. Test with this solvent was undertaken to ensure no solvent effect. Results showed that neutral red retention time (NRRT) measured at the end of the bioassay was significantly (p< 0.05) reduced (50 %) (p < 0.05) when exposed to environmental concentrations of pharmaceuticals (caffeine = 15 µg L-1; ibuprofen = 5 µg L-1; carbamazepine =1 µg L-1 and novobiocin = 10 µg L-1). Results showed dose-dependent effect of pharmaceuticals (p< 0.05). Clams analyzed after exposure to 10, 15 and 50 µg L-1 were considered to present a diminished health status (NRTT < 45 min) (p < 0.05). LMS appears to be a sensitive tool that enables evaluation of the health status of clams after exposure to concentrations of selected pharmaceuticals under laboratory conditions and could thus be used as a biomarker for pharmaceutical contamination in aquatic environments.